• International Journal of Stem Cells
• /
• v.16 no.2
• /
• pp.234-243
• /
• 2023

The recent advances in human pluripotent stem cells (hPSCs) enable to precisely edit the desired bases in hPSCs to be used for the establishment of isogenic disease models and autologous ex vivo cell therapy. The knock-in approach based on the homologous directed repair with Cas9 endonuclease, causing DNA double-strand breaks (DSBs), produces not only insertion and deletion (indel) mutations but also deleterious large deletions. On the contrary, due to the lack of Cas9 endonuclease activity, base editors (BEs) such as adenine base editor (ABE) and cytosine base editor (CBE) allow precise base substitution by conjugated deaminase activity, free from DSB formation. Despite the limitation of BEs in transition substitution, precise base editing by BEs with no massive off-targets is suggested to be a prospective alternative in hPSCs for clinical applications. Considering the unique cellular characteristics of hPSCs, a few points should be considered. Herein, we describe an updated and optimized protocol for base editing in hPSCs. We also describe an improved methodology for CBE-based C to T substitutions, which are generally lower than A to G substitutions in hPSCs.

• Genomics & Informatics
• /
• v.6 no.3
• /
• pp.157-159
• /
• 2008

Multiple sequence alignment (MSA) is the most important step for many of biological sequence analyses, homology search, and protein structural assignments. However, large amount of data make biologists difficult to perform MSA analyses and it requires much computational time to align many sequences. Here, we have developed a simple and fast web alignment tool for aligning, editing, and visualizing large amount of sequence data. We used a cluster server installed ClustalW-MPI using web services and message passing interface (MPI). It also enables users to edit multiple sequence alignments for manual editing and to download the input data and results such as alignments and phylogenetic tree.

• Animal Bioscience
• /
• v.36 no.2_spc
• /
• pp.333-338
• /
• 2023

Genetic modification enables modification of target genes or genome structure in livestock and experimental animals. These technologies have not only advanced bioscience but also improved agricultural productivity. To introduce a foreign transgene, the piggyBac transposon element/transposase system could be used for production of transgenic animals and specific target protein-expressing animal cells. In addition, the clustered regularly interspaced short palindromic repeat-CRISPR associated protein 9 (CRISPR-Cas9) system have been utilized to generate chickens with knockout of G0/G1 switch gene 2 (G0S2) and myostatin, which are related to lipid deposition and muscle growth, respectively. These experimental chickens could be the invaluable genetic resources to investigate the regulatory pathways and mechanisms of improvement of economic traits such as fat quantity and growth. The gene-edited animals could also be applicable to the livestock industry.

• Journal of Microbiology and Biotechnology
• /
• v.33 no.10
• /
• pp.1370-1375
• /
• 2023

In this study, we aimed to enhance the accumulation of chorismate (CHR) and anthranilate (ANT), key intermediates in the shikimate pathway, by modifying a shikimate over-producing recombinant strain of Corynebacterium glutamicum [19]. To achieve this, we utilized a CRISPR-driven genome engineering approach to compensate for the deletion of shikimate kinase (AroK) as well as ANT synthases (TrpEG) and ANT phosphoribosyltransferase (TrpD). In addition, we inhibited the CHR metabolic pathway to induce CHR accumulation. Further, to optimize the shikimate pathway, we overexpressed feedback inhibition-resistant Escherichia coli AroG and AroH genes, as well as C. glutamicum AroF and AroB genes. We also overexpressed QsuC and substituted shikimate dehydrogenase (AroE). In parallel, we optimized the carbon metabolism pathway by deleting the gntR family transcriptional regulator (IolR) and overexpressing polyphosphate/ATP-dependent glucokinase (PpgK) and glucose kinase (Glk). Moreover, acetate kinase (Ack) and phosphotransacetylase (Pta) were eliminated. Through our CRISPR-driven genome re-design approach, we successfully generated C. glutamicum cell factories capable of producing up to 0.48 g/l and 0.9 g/l of CHR and ANT in 1.3 ml miniature culture systems, respectively. These findings highlight the efficacy of our rational cell factory design strategy in C. glutamicum, which provides a robust platform technology for developing high-producing strains that synthesize valuable aromatic compounds, particularly those derived from the shikimate pathway metabolites.

• KSBB Journal
• /
• v.31 no.4
• /
• pp.193-199
• /
• 2016

Global warming caused from the heavy consumption of fossil fuel is one of the biggest problems to be solved. Biofuel has been gained more attention as an alternative to reduce the consumption of fossil fuel. Recently, butanol produced from the genus Clostridium has been considered as one of the promising alternatives for gasoline, fossil based fuel. Nevertheless, the lack of the genome-engineering tools for the genus Clostridium is the major hurdle for the economic production of butanol. More recently, genome engineering tools have been developed for metabolic engineering of butanol-producing Clostridium species, which includes genome scale network model and genome editing tools on the basis of mobile group II introns and CRISPR/Cas system. In this study, the genome engineering tools for butanol-producing Clostridium species have been reviewed with a brief future perspective.

• Journal of Life Science
• /
• v.29 no.5
• /
• pp.537-544
• /
• 2019

Genome editing technology employing gene scissors has generated interest in molecular breeding in various fields, and the development of the third-generation gene scissors of the clustered, regularly interspaced short palindromic repeat (CRISPR) system has accelerated the field of molecular breeding through genome editing. In this study, we analyzed the possibility of silkworm molecular breeding using gene scissors by genomic and phenotypic analysis after editing the biogenesis of lysosome-related organelles (BmBLOS) gene of Bakokjam using the CRISPR/Cas9 system. Three types of guide RNAs (gRNA) were synthesized based on the BmBLOS gene sequence of Bakokjam. Complexes of the prepared gRNA and Cas9 protein were formed and introduced into Bombyx mori BM-N cells by electroporation. Analysis of the gene editing efficiency by T7 endonuclease I analysis revealed that the B4N gRNA showed the best efficiency. The silkworm genome was edited by microinjecting the Cas9/B4N gRNA complex into silkworm early embryos and raising the silkworms after hatching. The hatching rate was as low as 18%, but the incidence of mutation was over 40%. In addition, phenotypic changes were observed in about 70% of the G0 generation silkworms. Sequence analysis showed that the BmBLOS gene appeared to be a heterozygote carrying the wild-type and mutation in most individuals, and the genotype of the BmBLOS gene was also different in all individuals. These results suggest that although the possibility of silkworm molecular breeding using the CRISPR/Cas9 system would be very high, continued research on breeding and screening methods will be necessary to improve gene editing efficiency and to obtain homozygotes.

• Korean Journal of Environmental Agriculture
• /
• v.40 no.3
• /
• pp.179-185
• /
• 2021

BACKGROUND: Tomato genome editing using CRISPR-Cas9 is being actively conducted in recent days, and lots of plant researches have been aiming to develop high valued crops by editing target genes without inserting foreign genes. Many researchers have been involved in the manipulation of the crop ripening process because fruit ripening is an important fruit phenotype for increasing fruit shelf life, taste, and texture of crops. This paper intends to evaluate target sgRNA to edit the two ripening-related genes encoding pectate lyase (PL) and phytoene synthase (Psy) with the CRISPR-Cas9 system. METHODS AND RESULTS: The CRISPR-Cas9 expression vector was cloned to target the PL (Solyc03g111690), Psy1 (Solyc03g031860), and Psy2 (Solyc02g081330) genes, which are the ripening genes of tomatoes. Tomatoes injected with Agrobacterium containing the CRISPR-Cas9 expression vector were further cultured for 5 days and used to check gene editing efficiency. As a result of the target gene sequence analysis by the next generation sequencing method, gene editing efficiency was calculated, and the efficient target location was selected for the PL and Psy genes. CONCLUSION: Therefore, this study was aimed to establish target sgRNA data that could have higher efficiency of the CRISPR-Cas9 system to obtain the delayed ripening phenotype of tomato. The developed method and sgRNA information is expected to be utilized in the development of various crops to manage its ripening processes.

• BMB Reports
• /
• v.49 no.4
• /
• pp.201-207
• /
• 2016

The CRISPR-Cas system has emerged as a fascinating and important genome editing tool. It is now widely used in biology, biotechnology, and biomedical research in both academic and industrial settings. To improve the specificity and efficiency of Cas nucleases and to extend the applications of these systems for other areas of research, an understanding of their precise working mechanisms is crucial. In this review, we summarize current studies on the molecular structures and dynamic functions of type I and type II Cas nucleases, with a focus on target DNA searching and cleavage processes as revealed by single-molecule observations.

• Genomics & Informatics
• /
• v.5 no.1
• /
• pp.30-31
• /
• 2007

Many visualization tools have been designed to aid information processing during whole genome projects. We have developed a trace viewer program, ChroView, which can read a chromatogram file and display the chromatogram traces of the four bases. The program can be used to examine sequencing quality and base-calling errors. It can also help researchers to edit and save base-calling results while browsing the traces. Additionally, this program has a basecalling feature which can produce supplementary data for validation of the results from other base-calling programs.

• Genomics & Informatics
• /
• v.18 no.4
• /
• pp.46.1-46.4
• /
• 2020

We developed the BaSDAS (Barcode-Seq Data Analysis System), a GUI-based pooled knockout screening data analysis system, to facilitate the analysis of pooled knockout screen data easily and effectively by researchers with limited bioinformatics skills. The BaSDAS supports the analysis of various pooled screening libraries, including yeast, human, and mouse libraries, and provides many useful statistical and visualization functions with a user-friendly web interface for convenience. We expect that BaSDAS will be a useful tool for the analysis of genome-wide screening data and will support the development of novel drugs based on functional genomics information.